I have isolated total protein from E.coli cells and seperated its soluble and insoluble fractions and run in SDS PAGE gel. After destaing i could see similar pattern (an size) of bands in both the supernatant and pellet fractions. Is there anything wrong?
Hei
My first answer: NO. shouldnt look similar ;)
But, the questions:
- what buffer(s) do you use for protein extraction?
- Of course there will be an overlap!
I now recognize that electrophoresis (SDS-PAGE, IEF, CE) is not suitable to separate the hydrophobic proteins, which will aggregate during separation due to strong Van Der Waals force. Thus, HPLC-SEC separation method is accordingly proposed.
"Hayakawa K, Yoshinaga T, Hirano M, Yoshikawa K, Katsumata N, Tanaka T, Nagamine T. Protein determination by high-performance gel-permeation chromatography: applications to human pancreatic juice, human bile and tissue homogenate. J Chromatogr B 754: 65-76, 2001."
"Hayakawa K, Guo L, Terentyeva EA, Li XK, Kimura H, Hirano M, Yoshikawa K, Yoshinaga T, Nagamine T, Katsumata N, Tanaka T. Size-exclusion chromatography of biological samples which contain extremely alkaline proteins. J Biochem Biophys Methods 56: 153-163, 2003."
This method using 1% non-ionic detergent seems powerful to me to separate the hydrophobic proteins in the state-of-the-art manner..
Your protein may have an association with membranes and thereby partition between both soluble and insoluble fractions.
As far as total protein is concerned, they often look similar (not identical), especially if you did not wash the pellet extensively (and if your goal is to produce a soluble recombinant protein, you probably didn't)).
Thank you all for your kind suggestions. Mr. Jonas Grossmann, I used TrisHCl buffer containing 1 mM DTT, 10% glycerol, 150 mM NaCl, and 0.1 mM EDTA supplemented with lysozyme.
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